Molybdenum, Tungsten, Vanadium, and Chromium - Biological Inorganic Chemistry - page 335

4S] 2 þ cluster is reduced back to the 1

replaced by MgATP, and the [4Fe oxidation state. Repetition of this cycle

of association, reduction, ATP hydrolysis, and dissociation transfers one electron at a time to the MoFe protein.
In the eight-state MoFe protein cycle, the MoFe protein is reduced successively by one electron, with the eight
states represented by E n . Usually, when 8 reducing equivalents have been accumulated, and 16 molecules of ATP
hydrolysed, the enzyme can bind and reduce the very stable triple bond of a dinitrogen molecule to two molecules
of ammonia. Concomitantly, two protons and two electrons are converted to gaseous hydrogen. Electrons derived
from photosynthesis or from the mitochondrial electron transport chain are transferred to the Fe-protein.
The structure of both the MoFe-cofactor and of the P-cluster became apparent when the structures of nitro-
genases were determined by high-resolution X-ray crystallography. The MoFe-cofactor ( Figure 17.13 a ) consists
of a [4Fe

e þ

3S] cluster by a previously undetected central atom (possibly
a nitrogen) at one corner and three bridging inorganic sulfides. (R)-homocitrate is coordinated to the Mo atom
through its 2-hydroxy and 2-carboxyl groups. The MoFe-cofactor is linked to the protein by only two residues,
Cys

e 3S] cluster connected to a [3Fe e Mo e

442, which coordinate Fe1 and the Mo atom respectively, at opposite ends of the extended
cluster. This is in marked contrast to other iron

a 273 and His a

sulfur clusters, which typically have one protein side-chain ligand
per metal ion. In order to complete the coordination sphere of the eight metal centres, there are a number of
additional inorganic sulfides together with bidentate coordination of the Mo atom to a molecule of homocitrate, 4
completing its octahedral coordination.
In the dithionite-reduced state ( Figure 17.13 ( b)) P N , the P-cluster can be considered as two [4Fe

e

3S] clusters
bridged by a hexacoordinate sulfur. In the P Ox state, which is oxidised by two electrons relative to P N , two of the
iron atoms Fe5 and Fe6 have moved away from the central sulfur atom, and are now coordinated by the amide
nitrogen of Cys

e

a a

186, maintaining the irons in a four-coordinate state.
The Fe-protein has the protein fold and nucleotide-binding domain of the G protein family of nucleotide-
dependent switch proteins, which are able to change their conformation dependent on whether a nucleoside
diphosphate (like GDP or ADP) is bound instead of the corresponding triphosphate (GTP or ATP). However,
nucleotide analogues which induce the conformational switch of the Fe-protein do not allow substrate reduction
by the MoFe protein, nor does reduction of the MoFe protein by other electron transfer reagents (whether small
proteins or redox dyes) drive substrate reduction. Only the Fe-protein can reduce the MoFe protein to a level that
allows it to reduce substrates like nitrogen.
Electrons arriving at the Fe-protein are transferred to the P-cluster and from there to the MoFe protein, which is
the site of interaction with dinitrogen or any of the other subrates which are reduced by nitrogenase. The redox
chemistry of nitrogen reduction, on the basis of model reactions first proposed by Chatt, involves nitrogenous
species at the level of diazene (N 2 H 2 ) and hydrazine (N 2 H 4 ) before the final release of two molecules of ammonia.
Recent evidence for a diazene-derived species bound to the FeMo-cofactor supports this view, as does evidence
that hydrazine (N 2 H 4 ) is a substrate for nitrogenase. A binding site for N 2 and for alkyne substrates has been
localised on the iron

87 and the hydroxyl of Ser

sulfur face of the FeMo-cofactor defined by the Fe atoms 2, 3, 6, and 7 ( Figure 17.13 (c)),
and ENDOR spectroscopy has shown that the alkene product of alkyne reduction is probably bound end-on to
a single Fe atom of the FeMo-cofactor.
A starting point for the nitrogenase reaction pathway can be proposed from the mechanisms of N 2 reduction
catalysed by organometallic complexes. A series of model studies initiated in the early 1960s by the groups of Chatt

e

=

FIGURE 17.13 (a) Structure of the FeMo-cofactor of nitrogenase. The element colours are as described in the legend to Figure 11 .
(b) P-cluster structures. Shown are the structures of the P-cluster [8Fe e 7S] in the oxidised (P ox ) and reduced (P N ) states. MoFe protein amino
acid ligands are also shown with b -188 Ser and a -88 Cys labelled. The central S atom is labelled S1. The PBD files used were 2MIN for the P ox state
and 3MIN for the P N state. (c) Substrate binding location on FeMo-cofactor. Shown is the FeMo-cofactor with Fe atoms 2, 3, 6, and 7 labelled.
The view is from the top looking down on the Fe face that binds substrates. Carbon alpha and the side chain are shown for a -69 Gly , a -70 Va l ,
a -195 His , and a -191 Gln . PDB file 1M1N. (From Seefeldt et al., 2009 . Copyright 2009, with permission from Annual reviews, Inc.)

4. A homologue of citrate (see Chapter 5) with an additional CH 2 group.